Role of interferon in natural kill of HSV-1-infected fibroblasts

The production of interferon during natural killer (NK) assays against HSV-1-infected fibroblasts (NK(HSV-1)) was studied to determine whether this interferon was responsible for inducing the preferential lysis of herpes-virus-infected target cells over uninfected target cells. The interferon produced during NK(HSV-1) assays was analyzed and found to have the properties of HU-IFN-alpha. Little or no IFN was produced during NK assays against uninfected fibroblasts (NK(FS)) or K562 (NK(K562)) cells. Although the appearance of interferon in the culture supernatants seemed to parallel the development of cytotoxicity during NK(HSV-1) assays, the levels of cytotoxicity and IFN generated did not correlate, arguing against a strict quantitative dependence of cytotoxicity upon IFN production. NK(K562) and NK(FS) cytotoxicity developed with little or no production of IFN. When IFN-pretreated effector cells were used, there was still a preferential lysis of infected over uninfected target cells. This preferential lysis by IFN-treated effector cells of infected over uninfected targets was seen as early as 2 hr into the assay. Anti-IFN antibodies added to the NK assays, although neutralizing all the IFN produced during the assays, had no effect on NK(FS) or NK(K562) cytotoxic activity and caused a slightly reduction of NK(HSV-1) activity only in one of three experiments. We conclude that although IFN is generated during NK(HSV-1) assays, this IFN cannot solely account for the increased lysis of infected over uninfected cells and that NK(HSV-1) activity is in some other way dependent on the virus infection.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Role of interferon in human natural killer activity against target cells infected with HSV-1

Human natural killer (NK) cells show high cytotoxic activity against target cells infected with herpes simplex virus type 1 (HSV-1). Substantial amounts of interferon (IFN) were generated in co-cultures of NK effector cells and infected target cells; however, the cytotoxic activity seen against a specific infected cell target did not correlate with the amount of IFN induced. The production of IFN increased steadily from 4 to 18 hr of co-culture, as did NK activity; however, IFN production peaked 4 hr later than NK activity. Pretreatment of NK effector cells with exogenous IFN increased cytotoxic activity against all targets tested, but the differential pattern of reactivity against cells infected with wild type and mutant viruses was unaltered. When effector cells were treated with the RNA synthesis inhibitor actinomycin D before co-culture with virus-infected targets, IFN production was markedly reduced, without a concomitant reduction in cytotoxicity. Similarly, the addition of anti-IFN antiserum to co-cultures greatly decreased the available IFN present, but had no effect on NK activity. We conclude that the induction of cytotoxic activity in co-cultures of NK effector cells and HSV-1-infected target cells is independent of the induction of IFN.     

Adenovirus E1A gene induction of susceptibility to lysis by natural killer cells and activated macrophages in infected rodent cells.

Rodent cells immortalized by the E1A gene of nononcogenic adenoviruses are susceptible to lysis by natural killer (NK) cells and activated macrophages. This cytolysis-susceptible phenotype may contribute to the rejection of adenovirus-transformed cells by immunocompetent animals. Such increased cytolytic susceptibility has also been observed with infected rodent cells. This infection model provided a means to study the role of E1A gene products in induction of cytolytic susceptibility without cell selection during transformation. Deletion mutations outside of the E1A gene had no effect on adenovirus type 2 (Ad2) or Ad5 induction of cytolytic susceptibility in infected hamster cells, while E1A-minus mutant viruses could not induce this phenotype. E1A mutant viruses that induced expression of either E1A 12S or 13S mRNA in infected cells were competent to induce cytolytic susceptibility. Furthermore, there was a correlation between the accumulation of E1A gene products in Ad5-infected cells and the level of susceptibility of such target cells to lysis by NK cells. The results of coinfection studies indicated that the E1A gene products of highly oncogenic Ad12 could not complement the lack of induction of cytolytic susceptibility by E1A-minus Ad5 virus in infected cells and also could not block induction of this infected-cell phenotype by Ad5. These data suggest that expression of the E1A gene of nononcogenic adenoviruses may cause the elimination of infected cells by the immunologically nonspecific host inflammatory cell response prior to cellular transformation. The lack of induction of this cytolysis-susceptible phenotype by Ad12 E1A may result in an increased persistence of Ad12-infected cells in vivo and may lead to an increased Ad12-transformed cell burden for the host.     

Downregulation of uridine-cytidine kinase like-1 decreases proliferation and enhances tumor susceptibility to lysis by apoptotic agents and natural killer cells

Natural killer (NK) cells target and kill tumor cells by direct anti-tumor cytotoxicity. NK lytic-associated molecule (NKLAM) is a protein involved in this cytolytic function. Acting as an E3 ubiquitin ligase, NKLAM binds to and ubiquitinates a novel protein, uridine-cytidine kinase like-1 (UCKL-1), targeting it for degradation. However, UCKL-1’s function in tumor cell survival and NK cell cytotoxicity is unknown. UCKL-1’s homology to uridine kinases and over expression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. We propose that NKLAM and UCKL-1 interact in the tumor cell, where degradation of UCKL-1 leads to increased tumor cell apoptosis. Here we use RNA interference to downregulate UCKL-1 expression in K562 erythroleukemia cells. It was seen that downregulation of UCKL-1 initiated apoptosis and slowed the cell cycle, resulting in lower growth in the small interfering UCKL-1 RNA treated K562 cell culture. In addition, the chemotherapeutic agent staurosporine was seen to be more effective in inducing cell death by apoptosis in UCKL-1 depleted K562 cells compared with controls. We also found that UCKL-1 depleted K562 cells were more susceptible to NK mediated cytolysis than controls. These results indicate a role for UCKL-1 in tumor cell survival and suggest possible therapeutic potential of UCKL-1 inhibitors in cancer treatment.     

Role of tumor necrosis factor-alpha in E1A oncogene-induced susceptibility of neoplastic cells to lysis by natural killer cells and activated macrophages

NIH-3T3 cells transfected with adenovirus E1A oncogene cDNA were found to exhibit cytolytic susceptibility to murine NK cells and activated macrophages associated with a threshold level of oncogene product expression exceeding that required for morphological transformation. A similar correlation was observed between threshold levels of E1A gene product expression and target cell susceptibility to direct cytotoxicity by rTNF. Inhibition of splenic NK cell and peritoneal macrophage cytolysis by antisera specific for murine rTNF confirmed the importance of E1A-induced TNF susceptibility as one determinant of target cell cytolytic susceptibility. Anti-TNF antibody was, however, unable to block killing of E1A-expressing targets by the NK cell line, NKB61A2. These results suggest a direct link between the functions of E1A oncogene products and cellular mechanisms of action of TNF elaborated by host effector cells and indicate that E1A expression also affects target cell susceptibility to TNF-independent cytolytic mechanisms.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.     

Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells

Background

Circulating CD34⁺ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN) could target progenitor cell injury by Natural Killer (NK) cells, thereby limiting their availability for vascular repair.

Methodology/Principal Findings

We show that CD34⁺-derived Endothelial Colony Forming Cells (ECFC) can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34⁺ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34⁺ cells expressing FKN was identified as an independent variable inversely correlated to CD34⁺ progenitor cell count. We further showed that treatment of CD34⁺ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression.

Conclusions

Our data highlights a novel mechanism by which FKN expression on CD34⁺ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.     

Tumor cell differentiation modulates susceptibility to natural killer cells

Induced differentiation in three human cell lines altered their sensitivity specifically to human natural killer (NK) cells by affecting their expression of NK target antigens. Differentiation of HL-60, a promyelocytic leukemia cell line, and the erythroleukemic cell line K562 was accompanied by a concomitant decrease in susceptibility to NK-mediated lysis whereas induction of MeWo melanoma cells resulted in an enhanced sensitivity to lysis. Our findings suggest that target cell susceptibility to NK-mediated lysis may in part be dependent on the stage of differentiation of the tumor cell target.     

High expression of the c-myc oncogene renders melanoma cells prone to lysis by natural killer cells

NK cells kill a wide variety of tumor cells, but usually leave normal cells intact. It was earlier reported that low class I HLA expression can be one of the factors that render target cells relatively susceptible to NK lysis. In this contribution, we show that in human melanomas the class I HLA expression is down-modulated by high expression of transfected c-myc oncogenes. The extent of down-modulation depended on the level of c-myc expression in a dose-dependent way. Taken together, these data suggested to us that one of the results of high c-myc expression could be the induction of a NK susceptible phenotype in melanoma cells. Therefore, we analyzed the effect of c-myc on NK susceptibility. We have found that high expression of transfected c-myc genes indeed converts the melanoma cell lines from poor into good targets for NK cells. IFN-gamma was used to restore the class I HLA expression of the c-myc transfectants, and the resulting cells showed a decreased NK susceptibility. These results suggest the possibility that the c-myc-induced NK susceptibility is mediated by the reduction of class I HLA expression.     

Inability of interferon to protect virus-infected cells against lysis by natural killer (NK) cells correlates with NK cell-mediated antiviral effects in vivo

Murine cytomegalovirus (MCMV) is a natural killer (NK) cell-sensitive virus, whereas lymphocytic choriomeningitis virus (LCMV) is an NK cell-resistant virus. Selective depletion of NK cell activity by injection of mice with anti-asialo GM1 antibody enhanced synthesis of MCMV but not that of LCMV when mice were simultaneously infected with the two viruses. This suggests that the NK cell-mediated antiviral effects may depend on target cell susceptibility to NK cell-mediated lysis rather than the ability of a virus to induce a specialized antiviral NK cell. In support of this concept, activated NK cells isolated from either MCMV- or LCMV-infected mice had similar patterns of killing against all targets tested. Mouse embryonic fibroblasts (MEF) infected with MCMV were less sensitive to lysis by activated NK cells than either uninfected or LCMV-infected MEF. However, when MEF were pretreated with IFN, activated NK cell-mediated lysis against MCMV-infected MEF was undiminished and was much higher (up to fourfold) than that against uninfected MEF, whose sensitivity to lysis was almost totally abolished by IFN pretreatment. LCMV-infected MEF were also protected by IFN against activated NK cell-mediated lysis. During infection, the virus-induced IFN may protect uninfected and LCMV-infected cells from IFN-activated, NK cell-mediated lysis, but MCMV-infected cells may remain sensitive to lysis. This could explain how NK cells play a role in resistance to MCMV but not LCMV.     

Oncogenicity of human papillomavirus- or adenovirus-transformed cells correlates with resistance to lysis by natural killer cells.

The reasons for the dissimilar oncogenicities of human adenoviruses and human papillomaviruses (HPV) in humans are unknown but may relate to differences in the capacities of the E1A and E7 proteins to target cells for rejection by the host natural killer (NK) cell response. As one test of this hypothesis, we compared the abilities of E1A- and E7-expressing human fibroblastic or keratinocyte-derived human cells to be selectively killed by either unstimulated or interferon (IFN)-activated NK cells. Cells expressing the E1A oncoprotein were selectively killed by unstimulated NK cells, while the same parental cells but expressing the HPV type 16 (HPV-16) or HPV-18 E7 oncoprotein were resistant to NK cell lysis. The ability of IFN-activated NK cells to selectively kill virally transformed cells depends on IFN's ability to induce resistance to NK cell lysis in normal (i.e., non-viral oncogene-expressing) but not virally transformed cells. E1A blocked IFN's induction of cytolytic resistance, resulting in the selective lysis of adenovirus-transformed cells by IFN-activated NK cells. The extent of IFN-induced NK cell killing of E1A-expressing cells was proportional to the level of E1A expression and correlated with the ability of E1A to block IFN-stimulated gene expression in target cells. In contrast, E7 blocked neither IFN-stimulated gene expression nor IFN's induction of cytolytic resistance, thereby precluding the selective lysis of HPV-transformed cells by IFN-activated NK cells. In conclusion, E1A expression marks cells for destruction by the host NK cell response, whereas the E7 oncoprotein lacks this activity.     

Degranulation of natural killer cells following interaction with HIV-1-infected cells is hindered by downmodulation of NTB-A by Vpu

Natural killer (NK) cell degranulation in response to virus-infected cells is triggered by interactions between invariant NK cell surface receptors and their ligands on target cells. Although HIV-1 Vpr induces expression of ligands for NK cell activation receptor, NKG2D, on infected cells, this is not sufficient to promote lytic granule release. We show that triggering the NK cell coactivation receptor NK-T- and -B cell antigen (NTB-A) alongside NKG2D promotes NK cell degranulation. Normally, NK cell surface NTB-A binds to NTB-A on CD4+ T cells. However, HIV-1 Vpu downmodulates NTB-A on infected T cells. Vpu associates with NTB-A through its transmembrane region without promoting NTB-A degradation. Cells infected with HIV-1 Vpu mutant elicited at least 50% more NK cells to degranulate than wild-type virus. Moreover, NK cells have a higher capacity to lyse HIV-infected cells with a mutant Vpu. Thus, Vpu downmodulation of NTB-A protects the infected cell from lysis by NK cells.     

Target cell susceptibility to lysis by human natural killer cells is augmented by alpha(1,3)-galactosyltransferase and reduced by alpha(1, 2)-fucosyltransferase

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha(1,3)Gal beta(1,4)GlcNAc [corrected], the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the alpha(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

N-acetylcysteine reduces transformed 3T3-SV40 fibroblast sensitivity to lysis by natural killer cells

We have shown that 18-20 h cultivation of transformed mouse fibroblasts 3T3-SV40 in the presence of antioxidant, N-acetylcysteine (NAC, 10 mM), did not change their sensitivity to lysis by natural killer (NK) mouse splenocytes. However, in 18-20 h after NAC removal 3T3-SV40 cells demonstrated resistance to NK cell activity. The cytotoxicity index (CI) was reduced up to 4.6 +/- 2.4 % (in comparison with the control value 31.8 +/- 2.4 %) approximating to the value in non-transformed 3T3 mouse fibroblasts. Normal 3T3 cells were resistant to NK action in all experimental conditions (CI varied within 0.7-5.3 %). These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect may be due to the NAC-induced modifications of the cell surface and extracellular matrix proteins.     

Recombinant gamma interferon enhances natural killer cell activity similar to natural gamma interferon

The enhancement of human natural killing activity by recombinant human gamma interferon (IFNγ) and natural human IFNγ were similar over a wide concentration range. Enhancement of natural killing activity by both interferons was neutralizable by antibody to natural IFNγ, as well as by antibody to a synthetic peptide representing the first 20 N-terminal amino acids of IFNγ provide conclusive evidence that IFNγ is responsible for the enhanced natural killing activity seen in IFNγ preparations.     

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.     

Immediate-early gene expression is sufficient for induction of natural killer cell-mediated lysis of herpes simplex virus type 1-infected fibroblasts.

Herpes simplex virus type 1 (HSV-1)-infected human fibroblast (HSV-FS) targets are susceptible to lysis by natural killer (NK) cells, whereas uninfected FS are resistant to lysis. Studies were undertaken to determine the mechanism of this preferential susceptibility. HSV-FS were not intrinsically less stable than FS, as determined by a 51Cr release assay under hypotonic shock in the presence of rat granule cytolysin and by sensitivity to anti-human leukocyte antigen class I antibody plus complement. Single-cell assays in agarose demonstrated that although similar numbers of large granular lymphocytes bound to the HSV-FS and FS targets, the conjugates with HSV-FS were lysed at a much higher frequency than those with FS. These results suggested that both targets are bound by the NK cells but only the HSV-FS were able to trigger lysis. The requirement for active virus expression was demonstrated by failure of emetine-treated HSV-FS targets or targets infected with UV-inactivated HSV to be lysed by NK effectors. To evaluate the role of viral glycoproteins in conferring susceptibility to lysis, Fab were prepared from HSV-1-seropositive sera; these Fab were unable to block lysis of the HSV-FS. Furthermore, incubation in phosphonoacetic acid failed to reduce NK(HSV-FS) activity despite sharp reductions in viral glycoprotein synthesis. Finally, targets infected with tsLB2 at the nonpermissive temperature were lysed as well as or better than targets infected with wild-type virus, indicating that HSV immediate-early gene product expression is sufficient for conferring susceptibility to lysis. We conclude that expression of nonstructural viral proteins or virally induced cellular gene products early in the course of infection rather than structural glycoproteins is required for NK lysis of HSV-FS targets.